Abstract
In pulse-chase experiments we compared the kinetics of early carbohydrate processing and subsequent secretion of thyroid-stimulating hormone (TSH) and free alpha subunit under control conditions and after treatment with 1-deoxynojirimycin, an inhibitor of glucosidases I and II. Under control conditions TSH achieved resistance to endo-beta-N-acetylglucosaminidase H (endo H) more rapidly than free alpha (t1/2 0.3 h versus 0.9 h); however, free alpha was secreted more rapidly than TSH (t1/2 2.2 h versus 3.4 h). With 1-deoxynojirimycin, oligosaccharides co-migrating with G3Man9GlcNAc and G2Man9GlcNAc were demonstrated on TSH for the first time, suggesting that previous pulse-chase studies did not disclose these intermediates due to rapid removal of glucose residues from the common G3Man9GlcNAc2 precursor. 1-Deoxynojirimycin delayed the rate of attainment of endo H resistance for both TSH and free alpha, but there was no effect on subunit combination. With 5 mM 1-deoxynojirimycin the amount of secreted free alpha was reduced to 65% of control; secreted TSH was reduced markedly to 17% of control without intracellular accumulation, suggesting increased intracellular degradation. There was no significant toxicity from these doses of 1-deoxynojirimycin on the production or secretion of the two major nonglycosylated pituitary proteins, growth hormone and prolactin, or on at least 10 other secretory proteins. Basal differences in the relative rates of TSH and free alpha processing and secretion as well as differential sensitivity to 1-deoxynojirimycin suggest separate secretory pathways for these two closely related proteins.
Highlights
In pulse-chase experiments we compared the kinetics the active hormone requires protein glycosylation and begins of early carbohydrate processing and subsequent se- in therough endoplasmic reticulum when the oligosaccharide cretion of thyroid-stimulating hormone (TSH)and free moiety is in the high-mannose form [5]
We have shown that treatment of mouse did not disclose these intermediates due to rapid re- thyrotropic tumorcells with tunicamycin, an agent that blocks moval of glucose residufersotmhe common asparagine-linked glycosylationcompletely [12], results in the GSManeGlcNAczprecursor.l-Deoxynojirimycin delayed the rate of attainment of endo H resistance for both TSH and freea,but there wasno effect on subunit combination
Inorder to study the early processing of TSHand free a in greater detail, dispersed mouse thyrotropic tumor cells were incubated in the presence of [3H]mannose for a 10-min pulse and chased for periods of 0, 30,60, and 120 min, which permitted analysis of possible differences in early processing
Summary
L-Deoxynojirimycin is a competitive antagonist to glucosidases I and I1 [13], the enzymes responsible for the initial removal of glucose residues from early high-mannose oligocin on the production or secretion of the two major saccharides of glycoproteins in the rough endoplasmic reticnonglycosylated pituitary proteins, growth hormone ulum. This agent is able to block or delay early high-mannose and prolactin, or on at least 10 other secretory proteins. They did not use specificinhibitors of carbohydrate processing enzymes to address the role of early processing on the sorting and secretion of the glycoprotein hormones.
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